Conventionally, true breeding and first filial generation (hereinafter referred to as F1) varieties have been generated as plant varieties. Among them, F1 varieties have become popular for staple crops. F1 varieties have major advantages including high growth rates, high yields, etc., as a result of high growing vigor due to hybrid vigor (heterosis). Additionally, they may be expected to have improvements in resistance to diseases and pest insects, and/or in adaptability to the environment, such as cold and heat resistance. Moreover, plants of an F1 variety have the same genotype, while they are heterozygous, and show very high uniformity in their phenotypes, increasing marketability of products from them. Furthermore, F1 varieties have increased probability of accumulating favorable traits that are governed by a dominant gene(s), enabling rapid breeding. Also, they provide a major advantage of protecting breeder's rights by raising the necessity of producing F1 seed every year for seed production, as their progeny will loose the uniformity in quality from the next generation as a result of segregation of the traits.
F1 varieties have become mainstream cultivated varieties for staple crops for the aforementioned reasons. For large scale production of F1 seed for commercial purposes, however, an economical and non-laborious method for emasculation is required. For plants that allow production of many seeds upon a single cross, including vegetables such as tomato, melon, cucumber and pumpkin, and flowering plants such as petunia and eustoma, economical production of F1 seed can be achieved by manual emasculation and crossing. However, there are still many crops that are very difficult to emasculate efficiently because of their flower structures. Crops that produce only a little amount of seed upon a single cross are difficult to produce hybrid seed in large quantities by manual crossing, making the production of F1 seed uneconomical. For seed production of such crops, it is crucial to develop a method using male sterility for seed production, because the laborious and expensive process of emasculation can be omitted by employing a male sterile plant as a seed parent, thus taking advantage of the genetic characteristics of male sterility.
Male sterility has been found in many plant species. And, methods for producing male sterile plants using protoplast fusion techniques have been established in many useful crops (Patent Documents 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19). There are various mechanisms for male sterility, which may be classified into two categories, the genetic male sterility and the cytoplasmic male sterility. Genetic male sterility is caused by a nuclear gene(s). On the other hand, cytoplasmic male sterility involves the interaction between a cytoplasmic genetic factor(s) and a nuclear gene(s).
Lettuce is produced in every country in the world. It is a vegetable of a very large market size, and the creation of its F1 varieties is highly desired. Non-patent Documents 1 and 2 describe the expression of heterosis in lettuce. The expression of heterosis is investigated by comparing F1 plants produced by using a genetic male sterile lettuce with conventional true breeds. At the conclusion of the investigation the investigators reported that the F1 plants exhibited outstanding heterosis. Consequently, a strong demand has been raised to establish a seed production technique using male sterility of lettuce to produce F1 seed efficiently. Yet such technique has not been established. For example, although Non-patent Document 3 revealed the mode of inheritance of genetic male sterility in lettuce, the practical application of genetic male sterility has remained problematic as it is unstable in male sterility and even female fertility appears to be defective (Non-patent Document 7). The author of Non-patent Document 3 also reported similar results in Non-patent Documents 4 and 5. Actually, while an F1 variety created by using genetic male sterility has been brought to market, it is unstable in male sterility and remains unable to replace the conventional true breed (Non-patent Document 6).
More recently, a novel type of genetic male sterile lines has been found. For example, the line “MS1024” described in Patent Document 1 and Non-patent Document 1 was found among progeny of plants selected for disease resistance. The line is superior to the genetic male sterile plants described in Non-patent Document 3 and the like in that it produces no pollen grain at all in its male sterility and that it provides a higher yield of seed.
However, the commercial F1 seed production using genetic male sterile plants still has a problem in the production cost. In lettuce, genetic male sterility results from homozygosis for a recessive gene in the cases observed so far (Non-patent Document 7). Because the male sterile plants resulted from homozygosis for a recessive gene are not able to reproduce by themselves, in order to maintain and reproduce a male sterile line it is necessary to cross them with individuals that are heterozygous for the male sterile gene, and screen their progeny for the individuals that are homozygous for the male sterile gene. Because the cross will result in 1:1 phenotypic segregation into fertile plant individuals and sterile plant individuals, about 50% of the hybrid progeny will be male sterile plant individuals. Therefore, for commercial F1 seed production the plants in seed farms must be screened to find and remove the fertile plant individuals in some way. In the case of lettuce this removing process is especially troublesome because it has small flower organs, making it difficult to distinguish sterile plant individuals and fertile plant individuals, and it produces many flowers on slender flower stalks.
Furthermore, the blooming stage is different for each individual so it is very laborious to remove fertile individuals completely based on the appearance of flowers, and the purity of F1 seed may be lowered by errors in the removing process, which may cause a problem by reducing the seed quality.
Additionally, breeding of genetic male sterile plants is inefficient and requires a long period of time because the individuals with the male sterile gene are visually indistinguishable from others except at the blooming stage. To facilitate the distinction between fertile plants and sterile plants, it is necessary to develop a method to select only the male sterile plants either by finding and making use of a gene that is linked to the morphology of seed in an early stage of growth, or by developing a DNA marker that is linked to the male sterile gene. Such a technique has not yet been established for lettuce, although studies for that are still under way.
Although it has been a long time since the mode of inheritance of genetic male sterility in lettuce was revealed, many problems in the development of F1 varieties using a genetic male sterile lettuce still remain unsolved as described above, and the creation of F1 varieties with high marketability is still difficult.
On the other hand, the production of F1 seed using cytoplasmic male sterility has been in practical use for a long time in sunflower, sugar beet, potato, rice, wheat, carrot, onion, and allium, etc., and commercial production systems for those crops have been established. Also in cole crops such as cabbage, broccoli, radish, and Chinese cabbage, in which F1 seed production using self-incompatibility has been widely used, use of cytoplasmic male sterility for F1 seed production has become popular recently due to the demand for seed of higher quality.
In general, once a cytoplasmic male sterile plant with stable male sterility is produced, cytoplasmic male sterility can be introduced into many desirable lines by repeated backcrossing and breeding of seed parents for F1 varieties becomes possible. Because cytoplasmic male sterility is transmitted by cytoplasmic inheritance, the progeny will be all male sterile. Cytoplasmic male sterile lines can be maintained and proliferated easily by crossing with a maintainer line that has the same nuclear genome and normal cytoplasm. Since there is no need of removing fertile individuals from seed parents in seed farms as in the seed production using genetic male sterility mentioned above, the seed production using cytoplasmic male sterility is efficient, and without the problem of the decreased purity of F1 seed due to errors in the removing process.
Based on these facts, a method for F1 seed production using a cytoplasmic male sterile plant is expected to be much more useful in an economical and commercial sense compared to those using genetic male sterility.
Despite the expectation that such a method would be highly useful as described above, a method for F1 seed production using a cytoplasmic male sterile plant in lettuce has not yet been developed. This is because, for lettuce, there is no crossable plant of the same species or genus that has cytoplasm with the ability to cause cytoplasmic male sterility when introduced into the cytoplasm of lettuce.
On the other hand, for sunflower cultivated species (Helianthus annuus L.) of the genus Helianthus of the family Asteraceae, many cytoplasmic male sterile plants have been found from the interspecific hybridization and F1 varieties are in practical use. Non-patent Documents 9-13 and other documents have reported on these. As an example of the production of a cytoplasmic male sterile vegetable in the family Asteraceae, it is known that cytoplasmic male sterile chicory can be produced by protoplast fusion of sunflower and chicory. Such techniques are disclosed, for example, in Patent Documents 2 and 3, and Non-patent Documents 14 to 17.
Non-patent document 14 was the first report of the production of cytoplasmic male sterile chicory, and revealed that cytoplasmic male sterile chicory can be produced by introducing cytoplasm of sunflower into chicory using the protoplast fusion technique. Non-patent documents 14 reported also that the mitochondria genome in the produced cytoplasmic male sterile chicory resulted from the recombination of the mitochondria genomes of chicory and sunflower. Furthermore, Non-patent Documents 16 and 17 reported the results of the analyses on the expression of cytoplasmic male sterility and on mitochondrial DNA structure in the progeny of the aforementioned cytoplasmic male sterile chicory.
Patent Document 2 describes a cytoplasmic male sterile plant of the family Asteraceae and a method for obtaining such a plant. The only cytoplasmic male sterile plant that was shown to be actually produced in Patent Document 2 is cytoplasmic male sterile chicory as in Non-patent Document. 14. Although Patent Document 2 refers to cytoplasmic male sterile lettuce, it only states methods for culturing and fusing protoplasts with sentences in the present tense, and neither method nor experiment result of the actual production of cybrid lettuce or cytoplasmic male sterile lettuce has been described. In the protoplast fusion technique, the process is relatively easy until the step of isolating and fusing protoplasts. For example, it is even possible to isolate and fuse plant protoplasts and animal cells. However, the steps of culturing the fused cells between phylogenetically distant species to have them divide, and further regenerate a plant are technically very difficult. This is because of the increased technical difficulties due to the stress during the fusing process, which may suppress the cell division of the fused cells, and the genetic incompatibility between the nuclei and cytoplasm from heterologous origins. To overcome these difficulties to regenerate a plant, it is necessary to develop a method for fusing cells without hurting their cell membrane, and a culturing method that allows the fused cells with new genomic combinations to divide and regenerate into a plant. Therefore, the techniques of culturing fused cells and subsequently regenerating a plant from the fused cells are the core techniques of the protoplast fusion technique. Furthermore, for having the expression of male sterility, the recombination or rearrangement of the mitochondrial genomes between different species in an adequate phylogenetic distance is necessary to occur. Thus, even after it becomes possible to produce a cybrid plant from two distantly related species, it is not predictable if it will be possible to produce a plant that expresses male sterility, and therefore, the step of actually producing cybrid plants and selecting an individual that exhibits male sterility is essential. Thus, crucial techniques to produce cytoplasmic male sterile lettuce are not disclosed in Patent Document 2. For these reasons, even after Patent Document 2 there are no reports of the successful production of cytoplasmic male sterile lettuce despite that there has been a strong demand for developing a method for producing cytoplasmic male sterile lettuce for a long time.
Patent Document 3 discloses a method of producing cytoplasmic male sterile chicory by introducing orf522, a gene that is thought to be a causal gene for cytoplasmic male sterility in sunflower, into a plant of the genus Cichorium by asymmetric protoplast fusion.
Non-patent Document 15 describes the production of cytoplasmic male sterile chicory by a new research group, which, in recent years, has been conducting studies to produce cytoplasmic male sterile chicory suitable for practical use, in continuation of the work.
As described above, there are many reports of the production of cytoplasmic male sterile chicory by introducing a part of sunflower mitochondrial DNA by protoplast fusion. However, there are no reports of the production of cytoplasmic male sterile lettuce in spite that lettuce and chicory are both in the family Asteraceae. The difficulty of producing cytoplasmic male sterile lettuce using cytoplasmic male sterile sunflower may be due to low genetic compatibility between the lettuce nuclear genome and the sunflower mitochondrial genome, compared to the genetic compatibility between the chicory nuclear genome and the sunflower mitochondrial genome.
Furthermore, one may consider introducing cytoplasmic male sterility into lettuce by crossing it with cytoplasmic male sterile chicory that is produced by introducing the sunflower cytoplasmic male sterile gene into chicory. However sexual hybridization between lettuce and chicory is difficult because they are in different genera and distantly related. There are no known reports of the successful production of cytoplasmic male sterile lettuce using such a method.
As described above, the production of cytoplasmic male sterile lettuce is technically very difficult, and it has not been successful yet. However the creation of F1 varieties of lettuce using cytoplasmic male sterile lettuce will be very beneficial. Therefore, the creation of cytoplasmic male sterile lettuce has been highly desired.
Patent Document 1 Japanese Patent Laid-Open No. 2005-110623
Patent Document 2 European Patent No. 0771523
Patent Document 3 International Publication No. WO97/45548
Patent Document 4 Japanese Patent Laid-Open No. 62-232324
Patent Document 5 Japanese Patent Laid-Open No. 63-79548
Patent Document 6 Japanese Patent Laid-Open No. 02-303426
Patent Document 7 Japanese Patent Laid-Open No. 63-36776
Patent Document 8 Japanese Patent Laid-Open No. 64-20041
Patent Document 9 Japanese Patent Laid-Open No. 01-218530
Patent Document 10 Japanese Patent Laid-Open No. 10-052185
Patent Document 11 U.S. Pat. No. 5,254,802
Patent Document 12 Japanese Patent Laid-Open No. 10-108676
Patent Document 13 Japanese Patent Laid-Open No. 10-108677
Patent Document 14 Japanese Patent Laid-Open No. 2001-145497
Patent Document 15 Japanese Patent Laid-Open No. 02-138927
Patent Document 16 Japanese Patent Laid-Open No. 01-206931
Patent Document 17 International Publication No. WO99/55143
Patent Document 18 International Publication No. WO95/09910
Patent Document 19 International Publication No. WO97/09873
Patent Document 20 U.S. Pat. No. 3,635,036
Non-patent Document 1 Takada, Katuya and Fujino, Masatake (1987) “Study on F1 seed production in Lettuce (1): Heterosis of F1 plants using male sterile lines as seed parents (in Japanese)” 1987 Spring Conference of Japanese Society for Horticultural Science, Research Abstract 208-209.
Non-patent Document 2 Takada, Katuya and Fujino, Masatake (1986) “Development of techniques using male sterility in lettuce (1): Expression of heterosis in F1 hybrids (in Japanese)” National Institute of Vegetable and Tea Science, Morioka Research Station, Annual Research Report No. 1, 87-93.
Non-patent Document 3 Edwrd J. Ryder (1967) A recessive male sterility gene in Lettuce (Lactica sativa L.) Pro. Am. Soc. Hortic. Sci. 91, 366-368.
Non-patent Document 4 Ryder, E, J Proceeding of the American society for horticultural Science 1963 Vol. 83585-589 An epistatically controlled pollen sterile in Lettuce (Lactica sativa L).
Non-patent Document 5 Ryder, E, J Science 1989 vol. 114(1) 129-133 Studies of three new genes, linkage, and epistasis in Lettuce.
Non-patent Document 6 Variety Registration Application “Fine” (in Japanese), Kaneko Seeds, No. 1745.
Non-patent Document 7 Serizawa, Hiroaki, “Recessive male sterile gene in lettuce (in Japanese)” Journal of Japanese Society for Horticultural Science, vol. 73, Supl. 2, p. 566.
Non-patent Document 8 Matsumoto, E., Plant cell reports 1991. vol. 9(10) Interspecific somatic hybridization between lettuce (Lactica sativa) and wild species L. virosa 
Non-patent Document 9 L. H. Rieseberg, C. Van Fossen, D. Arias, and R. L. Carter, The journal of heredity 1994: 85(3), 233-238 Cytoplasmic male sterility in Sunflower: origin, Inheritance, and Frequency in Natural Populations.
Non-patent Document 10 R. Horn Theor Appl Genet (2002) 104: 562-570 Molecular diversity of male sterility inducing and male-fertile cytoplasms in the genus Helianthus. 
Non-patent Document 11 S. Sukno, J. Ruso, Euphytica (1999) 106: 69-78 Interspecific hybridization between sunflower and wild perennial Herianthus species via embryo rescue.
Non-patent Document 12 R. Horn, W. Friedt, Plant Breeding 116 (1997) 317-322 Fertility restoration of new CMS sources in sunflower (Helianthus annuus L.)
Non-patent Document 13 Horn, R., Plant molecular biology; an international journal on fundamental research and genetic engineering July 1991 v17(1), 29-36 A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower.
Non-patent Document 14 C. Rambaud, J. Dubois, J. Vasseur (1993) Male-sterile chicory cybrids obtained by intergeneric protoplast fusion, Theor Appl Genet 87: 347-352
Non-patent Document 15 S. Varotto, E. Nenz, M. Lucchin, P. Parrini (2001) Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L., Theor Appl Genet 102: 950-956.
Non-patent Document 16 C. Rambaud, A. Bellamy, A. Dubreucq, J.-C. Bourquin and J. Vasseur (1997) Molecular analysis of the fourth progeny of plants derived from a cytoplasmic male sterile chicory cybrid, Plant Breeding 116: 481-486
Non-patent Document 17 A. Dubreucq, Theor Appl Genet (1999): 1094-1105 Analyses of mitochondrial DNA structure and expression in three cytoplasmic male-sterile chicories originating from somatic hybridization between fertile chicoly and CMS sunflower protoplasts.
Non-patent Document 18 Mizutani, Takayuki (1989) “Plant regeneration and protoplast fusion using protoplasts of lettuce and related Japanese wild species (in Japanese)” Bull. Fac. Agr., Saga Univ 67: 109-118.